E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

Ross, Wilma; Thompson, John F.; Newlands, Janet T.; Gourse, Richard L.

doi:10.1002/j.1460-2075.1990.tb07586.x

Cited by 360 publications

(451 citation statements)

References 53 publications

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“…1. ⌬72 indicates a deletion of an A:T base pair at position Ϫ72 at rrnB P1, which results in loss of binding and activation by the factor for inversion stimulation (36).…”

Section: Strainsmentioning

confidence: 99%

“…Multiple-round transcription reactions were performed at 30°C for 20 min using a previously described method (36). Each 25-l reaction mixture contained 40 mM Tris-acetate (pH 7.9), 150 mM KCl, 10 mM MgCl 2 , 1 mM dithiothreitol, 100 g/ml acetylated bovine serum albumin (Promega), 200 M ATP, 200 M GTP, 200 M CTP, 10 M UTP, and 5 Ci of [␣-32 P]UTP (800 Ci/mmol; Perkin-Elmer).…”

Section: Measurement Of Transcription In Vivomentioning

confidence: 99%

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The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

Husnain

Thomas

2008

J Bacteriol

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The Escherichia coli guaB promoter (P guaB ) regulates the transcription of two genes, guaB and guaA, that are required for de novo synthesis of GMP, a precursor for the synthesis of guanine nucleoside triphosphates. The activity of P guaB is subject to growth rate-dependent control (GRDC). Here we show that the A؉T-rich sequence located between positions ؊59 and ؊38 relative to the guaB transcription start site stimulates transcription from P guaB ϳ8-to 10-fold and, in common with other UP elements, requires the C-terminal domain of the RNA polymerase ␣ subunit for activity. Like the rrnB P1 UP element, the P guaB UP element contains two independently acting subsites located at positions ؊59 to ؊47 and ؊46 to ؊38 and can stimulate transcription when placed upstream of the lacP1 promoter. We reveal a novel role for the P guaB UP element by demonstrating that it is required for GRDC. The involvement of the UP element in GRDC also requires the participation of sequences located at least 100 bp upstream of the guaB transcription start site. These sequences are required for down-regulation of P guaB activity at lower growth rates.Escherichia coli RNA polymerase (RNAP) is a multisubunit complex consisting of an ␣ subunit homodimer, single ␤, ␤Ј, and subunits, and one of several subunits (14). The ␣ subunit contains an N-terminal domain (␣NTD; residues 8 to 232) and a C-terminal domain (␣CTD; residues 249 to 329) (5, 21, 44). ␣NTD and ␣CTD are separated by a flexible linker that is at least 13 amino acids long (5,20,25). ␣CTD plays important roles in transcription activation by contacting a number of transcription factors (6, 18) or through interacting with an AϩT-rich DNA sequence referred to as the UP element (16).UP elements stimulate transcription by recruiting ␣CTD to DNA (32, 43). The best-studied UP element spans the region from Ϫ59 to Ϫ38 at the rrnB P1 promoter (16). The rrnB P1 UP element contains two domains, a ϳ9-bp proximal UP element subsite located at positions Ϫ46 to Ϫ38 and a ϳ13-bp distal UP element subsite located at positions Ϫ59 to Ϫ47. Each subsite functions independently by recruiting one ␣CTD (11). The amino acid residues in ␣CTD that comprise the 265 determinant, including residues V264, R265, N268, N294, G296, K298, and S299, interact with UP element and A-tract sequences by contacting the DNA backbone, and R265 makes additional contacts with bases in the minor groove (4, 32, 43). These residues are the most important for rrnB P1 UP element utilization both in vivo and in vitro (13, 35). In the absence of a strong distal UP element subsite, ␣CTD bound to a consensus or near-consensus proximal UP element subsite also makes productive contacts with region 4.2 of 70 that serve to stimulate transcription (7,35).The E. coli guaB promoter (P guaB ) regulates the transcription of two genes, guaB and guaA, which together constitute the guaBA operon. The guaB and guaA genes encode IMP dehydrogenase and GMP synthetase, respectively, and are required for the biosynthesis of GMP from IMP (22, 42). P guaB...

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“…1. ⌬72 indicates a deletion of an A:T base pair at position Ϫ72 at rrnB P1, which results in loss of binding and activation by the factor for inversion stimulation (36).…”

Section: Strainsmentioning

confidence: 99%

Section: Measurement Of Transcription In Vivomentioning

confidence: 99%

The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

Husnain

Thomas

2008

J Bacteriol

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“…However, it cannot be predicted if Fis binding might occlude or enhance binding of other proteins at the ␥ origin. Prebinding of Fis at its own promoter excludes RNA polymerase (1a), but Fis binding enhances RNA polymerase binding elsewhere (62,68).…”

Section: Discussionmentioning

confidence: 99%

“…Escherichia coli RLG1679 is MG1655 ⌬lacX74 i 21 (rrnB P1-lac-lacZ) (68). Strain RLG1677 is RLG1679 fis::kan (31); this fis allele was obtained from R. Kahmann (46).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis

Ehley

et al. 1996

J Bacteriol

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Fis protein is shown here to bind to 10 sites in the ␥ origin of plasmid R6K. The Fis-binding sites overlap all the previously identified binding sites in the ␥ origin for the plasmid-encoded initiator protein and three host-encoded proteins, DnaA, integration host factor, and RNA polymerase. However, the requirement of Fis for R6K replication depends on the use of copy-up -protein variants and, oddly, the antibiotic resistance marker on the plasmid. In Fis-deficient cells, copy-up variants cannot drive replication of R6K ␥-origin plasmids carrying the bla gene encoding resistance to penicillin (Pen r ) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cm r ). In contrast, R6K replication driven by wild-type is unaffected by the antibiotic resistance marker in the absence of Fis protein. Individually, none of these elements (copy-up , Fis deficiency, or drug markers) prevents R6K replication. The replication defect is not caused by penicillin in the medium or runaway replication and is unaffected by the orientation of the bla gene relative to the origin. Replication remains inhibited when part of the bla coding segment is deleted but the bla promoter is left intact. However, replication is restored by insertion of transcriptional terminators on either side of the ␥ origin, suggesting that excess transcription from the bla gene may inactivate replication driven by copy-up mutants in the absence of Fis. This study suggests that vector sequences such as drug markers may not be inconsequential in replication studies, as is generally assumed.The small, abundant DNA-binding proteins Fis integration host factor (IHF), and HU (for reviews, see references 14, 24, 25, and 69) have roles in a variety of DNA transactions, including replication of many replicons. Fis binds and bends oriC, the origin of replication of the chromosome (22, 30), and is involved in chromosome replication (22). IHF also binds oriC (21, 63), and both HU and IHF assist DnaA in unwinding the origin (37) and participate in replication of oriC minichromosomes (21,42). IHF also has roles in the replication and stable maintenance of phage f1 (33, 34) and plasmids pSC101 (3-5, 27, 74, 75) and R6K (11-13, 15, 17, 45).Plasmid R6K encodes resistance to penicillin (Pen r ) and streptomycin (49) and contains three origins of replication: ␣, ␥, and ␤ (Fig. 1). Each origin requires in cis the 277-bp core segment and an additional segment unique to that origin (47,48,(70)(71)(72). The ␥ origin, for example, includes the core and the adjacent enhancer (for a recent review, see reference 16). The enhancer contains DnaA box 1 (79, 80) and a small segment, stb, involved in stable maintenance of ␥-origin-containing plasmids (81). We subdivided the core into three distinct regions (16): (i) the AT-rich region, bound by and IHF (11,13,15,17,19,45,52); (ii) seven 22-bp direct repeats (DRs) bound by (19,23,28,60); and (iii) a multiprotein-binding region interacting with D...

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“…A promoter proximal region, the UP element, is a cis-acting DNA sequence located between -60 and -40 with respect to the transcription start site which interacts directly with the C-terminal portion of the a subunit of RNA polymerase and increases transcription about 30-fold (2-6 and W.Ross, E.Blatter, R.H.Ebright, and R.L.Gourse, unpublished results). Furthermore, a promoter distal region, located between -150 and -60, increases transcription about 10-fold by binding the FIS protein at three sites (7). FIS Site I, the site closest to the promoter, accounts for 70 -80% of the effect of FIS on transcription (7).…”

Section: Introductionmentioning

confidence: 99%

Localization of the intrinsically bent DNA region upstream of theE.coli rrnBP1 promoter

Gaál¹,

Lin²,

Estrem³

et al. 1994

Nucl Acids Res

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DNA sequences upstream of the rrnB P1 core promoter (-10, -35 region) increase transcription more than 300-fold In vivo and in vitro. This stimulation results from a cis-acting DNA sequence, the UP element, which interacts directly with the alpha subunit of RNA polymerase, increasing transcription about 30-fold, and from a positively acting transcription factor, FIS, which increases expression another 10-fold. A DNA region exhibiting a high degree of intrinsic curvature has been observed upstream of the rrnB P1 core promoter and has thus been often cited as an example of the effect of bending on transcription. However, the precise position of the curvature has not been determined. We address here whether this bend is in fact related to activation of rRNA transcription. Electrophoretic analyses were used to localize the major bend in the rrnB P1 upstream region to position approximately -100 with respect to the transcription initiation site. Since most of the effect of upstream sequences on transcription results from DNA between the -35 hexamer and position -88, i.e. downstream of the bend center, these studies indicate that the curvature leading to the unusual electrophoretic behavior of the upstream region does not play a major role in activation of rRNA transcription. Minor deviations from normal electrophoretic behavior were associated with the region just upstream of the -35 hexamer and could conceivably influence interactions between the UP element and the alpha subunit of RNA polymerase.

show abstract

E.coli Fis protein activates ribosomal RNA transcription in vitro and in vivo.

Cited by 360 publications

References 53 publications

The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

The UP Element Is Necessary but Not Sufficient for Growth Rate-Dependent Control of the Escherichia coli guaB Promoter

Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis

Localization of the intrinsically bent DNA region upstream of theE.coli rrnBP1 promoter

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